Quantitative analysis basically involves three steps. First, use pure standard of the analyte to prepare calibration solutions at different concentration. Secondly, analyze the calibration solutions and unknown samples to obtain their signal responses. Finally, plot the signal strength (Y) measured from the calibration solutions samples against their concentrations (C) to establish a calibration curve. Concentration of the unknown samples can be calculated from the calibration equation or directly find from the curve. An ideal calibration curve is a straight line that fits a calibration equation Y=aC+b. Practically, linearity of a calibration curve only exists in a relatively small range, for example, between concentration 1 and 10. For a wide contraction range, a calibration curve is normally curved becoming a true “curve” that can better fit the equation Y=ax2+bx+c.
To ensure the accuracy of quantitative analysis, the general practice of the field is to implement the Internal Standard (IS) method. Draft of instrument sensitivity over time and erratic sample volume occurred during sample preparation are the major variations that cause inaccuracy in quantitative results. By accurately adding an internal standard, a pure substance (non-analyte), into each sample, then preparing the sample and measuring both the analyte and the internal standard during each analytical run, the variations can be largely cancelled out by taking the ratio of signal strength of the analyte (Ix) against that of the internal standard (Iis). When the analyte signal (Ix) rises or falls either due to change of sensitivity in the instrument or accidental error in sample volume, the internal standard signal would rise or fall accordingly, and the ratio of the signals (Ix/Iis) would remain unchanged. Therefore, for quantitative analysis using internal standard method, the calibration curve is the plot of the signal ratio (Y=Ix/Iis) against concentration (C). Since errors introduced during the sample processing are human error that could be avoided by caution and/or automation, internal standard method is most necessary for compensating instrument instability.
A mass spectrometer is a device for separation and detection of charged particles. Owing to its extreme sensitivity and unparalleled resolution, mass spectrometer has become a common tool for trace analysis. It is particularly indispensible for the studies of pharmacokinetics, drug metabolism, toxicology toxicokinetics, and clinical trials. When using a mass spectrometer for chemical analysis, the analyte has to be ionized first. Ionization of molecules is usually achieved in an ion source by high electric fields or particle bombardment. However, the stability of an electric field or bombast particle intensity in the ion source is never easy to maintain let alone the ion source is under constant input of a chromatographic effluent. Therefore, it is inevitable that determination sensitivity of mass spectrometry changes over time. Normally eight-hour variation an ion source can go beyond 30%. Thus, the accuracy of quantitative analysis with mass spectrometry without internal standard can only reach about 70%. In principle, internal standard method should be able to increase accuracy above 90%. However, the ionization efficiency is closely related to the structure of the substance, and the subtle differences in structure make the ionization efficiency to be very different. It is very difficult to find an internal standard substance whose ionization properties are consistent with those of the substance to be tested. At present, only the isotopologues of the analytes can be used as internal standards. Isotopologues are the replacement of certain elements in analytes with heavy isotopes, such as the replacement of hydrogen with deuterium, to obtain variant molecules that have higher molecular weight but structurally identical. When the analyte and such isotopologues are simultaneously introduced into the mass spectrometer to perform determination, the signals of the two can be distinguished due to the high resolution of the mass spectrometry. The ratio of their signal intensities is the basis of the quantification of the internal standard method. In summary, the only way to improve the quantitative accuracy of mass spectrometry at present is to use isotopologues as internal standards, which usually called isotope internal standards.
The current problems with mass spectrometry quantification are as follows: although isotope internal standards can increase the accuracy of mass spectrometry quantification from about 70% to more than 95%, which is comparable to the accuracy that can be achieved with other instruments, the cost of isotope internal standard synthesis is very high. At present, in addition to using this method in the analysis of clinical trials in the development of new drugs, most other applications, including preclinical experiments in the pharmaceutical process, cannot use this method due to high expenses and the time-consuming for the synthesis. This greatly hampers the widespread application of mass spectrometry technology, while also delaying the biopharmaceutical process and increasing costs. Moreover, if there is no problem that the internal standard is difficult to find, mass spectrometry technology can play a greater role in the field of clinical testing, environmental analysis, food safety and so on.
Therefore, the prior art still needs improvement and development.